Recombinant Anti-CD20 antibody [EP459Y] - Mouse IgG2a (Chimeric) (ab279299)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Mouse monoclonal [EP459Y] to CD20 - Mouse IgG2a
- Suitable for: ICC, Flow Cyt (Intra), IP, WB
- Knockout validated
- Reacts with: Human
Overview
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Product name
Anti-CD20 antibody [EP459Y] - Mouse IgG2a (Chimeric)
See all CD20 primary antibodies -
Description
Mouse monoclonal [EP459Y] to CD20 - Mouse IgG2a -
Host species
Mouse -
Tested applications
Suitable for: ICC, Flow Cyt (Intra), IP, WBmore details -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Raji and Ramos whole cell lysate and Wild-type Raji cell lysate ICC: Ramos cells. Flow Cyt (intra): Ramos cells. IP: Ramos whole cell lysate.
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General notes
This mouse monoclonal chimeric antibody has been engineered from a RabMAb parent antibody (ab78237). By necessity, some rabbit sequence is retained as part of the variable domain. When multiplexing with other rabbit-derived antibodies, using cross absorbed Fc-reactive secondary antibodies are recommended.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EP459Y -
Isotype
IgG2a -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab279299 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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ICC |
Use a concentration of 0.2 - 1 µg/ml.
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Flow Cyt (Intra) |
Use a concentration of 0.2 µg/ml.
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IP |
1/30.
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WB |
1/1000.
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Notes |
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ICC
Use a concentration of 0.2 - 1 µg/ml. |
Flow Cyt (Intra)
Use a concentration of 0.2 µg/ml. |
IP
1/30. |
WB
1/1000. |
Target
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Function
This protein may be involved in the regulation of B-cell activation and proliferation. -
Tissue specificity
Expressed on B-cells. -
Involvement in disease
Defects in MS4A1 are the cause of immunodeficiency common variable type 5 (CVID5) [MIM:613495]; also called antibody deficiency due to CD20 defect. CVID5 is a primary immunodeficiency characterized by antibody deficiency, hypogammaglobulinemia, recurrent bacterial infections and an inability to mount an antibody response to antigen. The defect results from a failure of B-cell differentiation and impaired secretion of immunoglobulins; the numbers of circulating B cells is usually in the normal range, but can be low. -
Sequence similarities
Belongs to the MS4A family. -
Post-translational
modificationsPhosphorylated. Might be functionally regulated by protein kinase(s). -
Cellular localization
Membrane. - Information by UniProt
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Database links
- Entrez Gene: 931 Human
- Omim: 112210 Human
- SwissProt: P11836 Human
- Unigene: 712553 Human
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Alternative names
- APY antibody
- ATOPY antibody
- B lymphocyte antigen CD20 antibody
see all
Images
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All lanes : Anti-CD20 antibody [EP459Y] - Mouse IgG2a (Chimeric) (ab279299) at 1/1000 dilution
Lane 1 : Wild-type Raji cell lysate
Lane 2 : MS4A1 knockout Raji cell lysate
Lane 3 : A549 cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 33 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-CD20 antibody [EP459Y] – Mouse IgG2a (Chimeric) staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab279299 was shown to bind specifically to CD20. A band was observed at 33 kDa in wild-type Raji cell lysates with no signal observed at this size in MS4A1 knockout cell line ab273871 (knockout cell lysate ab263259). To generate this image, wild-type and MS4A1 knockout Raji cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
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All lanes : Anti-CD20 antibody [EP459Y] - Mouse IgG2a (Chimeric) (ab279299) at 1/1000 dilution
Lane 1 : Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate
Lane 2 : Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution
Exposure time: 10 secondsBlocking/Dilution buffer: 5% NFDM/TBST.
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Immunofluorescence staining of CD20 using ab279299 in Ramos (human Burkitt's lymphoma cell line) cells.
The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab279299 at 0.2 µg/ml. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and nuclear DNA was labelled with DAPI (shown in blue).
The secondary only control (bottom row) was not incubated with ab279299 but otherwise processed the same.
Images were acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.
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Flow cytometry overlay histogram showing Ramos (human Burkitt's lymphoma cell line) positive cells (left panel) and negative HEK293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells (right panel) stained with ab279299 (red line).
The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab279299) (1x106 in 100µl at 0.2 µg/ml) for 30 min at 22°C.
The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150177) was used at 1/2000 for 30 min at 22°C.
Isotype control antibody (black line) was mouse IgG2a kappa (ab18413) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
This antibody gave a positive signal in Ramos cells fixed with 80% methanol (5 min) / permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.
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CD20 was immunoprecipitated from 0.35 mg Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate 10 µg with ab279299 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab279299 at 1/1000 dilution. mouse IgG for IP (HRP) (ab131368) was used at 1/5000 dilution.
Lane 1: Ramos whole cell lysate 10µg.
Lane 2: ab279299 IP in Ramos whole cell lysate.
Lane 3: Mouse monoclonal IgG2a (ab18413) instead of ab279299 in Ramos whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 5.5 seconds.
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Immunofluorescence staining of CD20 using ab279299 in Ramos (human Burkitt's lymphoma cell line) cells.
The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab279299 at 0.2 µg/ml. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and nuclear DNA was labelled with DAPI (shown in blue).
The secondary only control (bottom row) was not incubated with ab279299 but otherwise processed the same.
Images were acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab279299 has not yet been referenced specifically in any publications.